Published on Web 09/07/2006
Binding of a Designed Substrate Analogue to Diisopropyl Fluorophosphatase: Implications for the Phosphotriesterase Mechanism Marc-Michael Blum,† Frank Lo¨hr,† Andre Richardt,‡ Heinz Ru¨terjans,*,† and Julian C.-H. Chen*,† Contribution from the Institute of Biophysical Chemistry, J.W. Goethe UniVersity Frankfurt, Max-Von-Laue-Strasse 9, D-60438 Frankfurt, Germany, and Armed Forces Scientific Institute for Protection TechnologiessNBC Protection, D-29633 Munster, Germany Received March 20, 2006; E-mail:
[email protected];
[email protected] Abstract: A wide range of organophosphorus nerve agents, including Soman, Sarin, and Tabun is efficiently hydrolyzed by the phosphotriesterase enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. To date, the lack of available inhibitors of DFPase has limited studies on its mechanism. The de novo design, synthesis, and characterization of substrate analogues acting as competitive inhibitors of DFPase are reported. The 1.73 Å crystal structure of O,O-dicyclopentylphosphoroamidate (DcPPA) bound to DFPase shows a direct coordination of the phosphoryl oxygen by the catalytic calcium ion. The binding mode of this substrate analogue suggests a crucial role for electrostatics in the orientation of the ligand in the active site. This interpretation is further supported by the crystal structures of double mutants D229N/N120D and D229N/N175D, designed to reorient the electrostatic environment around the catalytic calcium. The structures show no differences in their calcium coordinating environment, although they are enzymatically inactive. Additional double mutants E21Q/N120D and E21Q/N175D are also inactive. On the basis of these crystal structures and kinetic and mutagenesis data as well as isotope labeling we propose a new mechanism for DFPase activity. Calcium coordinating residue D229, in concert with direct substrate activation by the metal ion, renders the phosphorus atom of the substrate susceptible for attack of water, through generation of a phosphoenzyme intermediate. Our proposed mechanism may be applicable to the structurally related enzyme paraoxonase (PON), a component of high-density lipoprotein (HDL).
Introduction
Chart 1
Diisopropyl fluorophosphatase from the squid Loligo Vulgaris (DFPase, EC 3.1.8.2, 35 kDa) efficiently detoxifies highly toxic organophosphorus compounds that act as suicide inhibitors of acetylcholinesterase. Besides the compound diisopropylfluorophosphate (DFP), the enzyme also detoxifies the range of G-type nerve agents including Tabun (GA), Sarin (GB), Soman (GD), and Cyclohexylsarin (GF)1-3 (Chart 1a). Detoxification is achieved by hydrolysis of the bond between phosphorus and the fluoride or cyanide leaving group. DFPase is remarkably stable and can be highly expressed, making it a top candidate for enzymatic decontamination of chemical warfare agents.4 According to the enzyme classification of the International Union of Biochemistry and Molecular Biology (IUBMB), organophosphorus-hydrolyzing enzymes are found within class EC 3.1.8 (phosphotriesterases), although these enzymes show wide biochemical and sequence diversity. As nerve agents are †
Institute of Biophysical Chemistry. ‡ Armed Forces Scientific Institute for Protection TechnologiessNBC Protection. (1) Hartleib, J.; Ruterjans, H. Protein Expression Purif. 2001, 21, 210-219. (2) Scharff, E. I.; Koepke, J.; Fritzsch, G.; Lucke, C.; Ruterjans, H. Structure (Cambridge, MA, U.S.) 2001, 9, 493-502. (3) Hoskin, F. C. G.; Roush, A. H. Science 1982, 215, 1255-1257. (4) Yang, Y. C.; Baker, J. A.; Ward, J. R. Chem. ReV. 1992, 92, 1729-1743. 12750
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(a) Substrates of DFPase. Nerve agents are indicated by their common names and U.S. two-letter abbreviations; (b) compounds screened for interaction with DFPase.
synthetic compounds, they are not the native substrates of these enzymes. Several enzymes with activities against toxic organophosphorus compounds have been reported but only DFPase from L. Vulgaris, organophosphorus hydrolase (OPH) from 10.1021/ja061887n CCC: $33.50 © 2006 American Chemical Society
Binding of a Designed Substrate Analogue to DFPase
ARTICLES
Figure 1. (a) Stereoview of DcPPA in the DFPase active site with Fo-Fc simulated annealing omit map, contoured at 2.5 σ, calculated in CNS by omitting the inhibitor from the structure factor calculation. (b) Interaction of the substrate analogue DcPPA with DFPase. Potential hydrogen bond distances are indicated in Å, as well as the catalytic calcium-phosphoryl oxygen distance. The figure was generated using PYMOL (http://pymol.sourceforge.net).
Pseudomonas diminuta,5,6 and organophosphorus acid anhydrase (OPAA) from Alteromonas7,8 can be expressed in kilogram quantities, are stable, and show the catalytic rate enhancements required for rapid decontamination. Interestingly, the mammalian enzyme paraoxonase (PON, EC 3.1.8.1), a high-density lipoprotein (HDL) component9 that may function to inactivate toxic byproducts of lipid oxidation by the low-density lipoprotein (LDL) complex, is structurally related to DFPase and shows activity against organophosphate triesters.10 Although the natural function of squid type DFPase and Pseudomonas OPH is unknown, the Alteromonas OPAA has the function of a prolidase (E.C. 3.4.13.9).11 Squid-type DFPase from L. Vulgaris, which is the subject of this work, was originally isolated from squid head ganglion. The enzyme consists of 314 amino acids and contains two calcium ions. DFPase has been overexpressed in E. coli and purified,1 and bulk quantities are readily available from expression in the yeast P. pastoris. Crystal structures of the DFPase apoenzyme have been solved at 1.8 Å and 0.85 Å resolution.2,12 The overall structure resembles a six-bladed pseudosymmetrical β-propeller with a central water filled tunnel. A high affinity calcium ion is located in the center of the molecule and has a structural role, while the second, low-affinity calcium ion is essential for catalytic activity and is located at the base of the active site, sealing the water filled tunnel (Figure 1a). The low affinity calcium ion is crucial for catalysis, as removal of this metal ion results in a folded, yet inactive enzyme.13 From the X-ray structure and mutagenesis experiments, H287 in the active site was postulated to be essential for catalysis, acting as a general base. On the basis of these findings, a reaction mechanism for DFPase was proposed. The substrate coordinates the calcium ion via the phosphoryl oxygen, such that the fluoride leaving group points away from H287. The histidine then activates water by proton abstraction, allowing for a backside attack of the nucleophile on the phosphorus. From the pentacoordinate transition state, a fluoride ion leaves as the first product followed by the phosphoric acid anion. However, (5) Munnecke, D. M. Appl. EnViron. Microb. 1976, 32, 7-13. (6) Dumas, D. P.; Caldwell, S. R.; Wild, J. R.; Raushel, F. M. J. Biol. Chem. 1989, 264, 19659-19665. (7) Defrank, J. J.; Cheng, T. C. J. Bacteriol. 1991, 173, 1938-1943. (8) Cheng, T. C.; DeFrank, J. J.; Rastogi, V. K. Chem.-Biol. Interact. 1999, 119-120, 455-462. (9) Gaidukov, L.; Tawfik, D. S. Biochemistry 2005, 44, 11843-11854. (10) Harel, M.; Aharoni, A.; Gaidukov, L.; Brumshtein, B.; Khersonsky, O.; Meged, R.; Dvir, H.; Ravelli, R. B.; McCarthy, A.; Toker, L.; Silman, I.; Sussman, J. L.; Tawfik, D. S. Nat. Struct. Mol. Biol. 2004, 11, 412-419. (11) Cheng, T. C.; Harvey, S. P.; Chen, G. L. Appl. EnViron. Microb. 1996, 62, 1636-1641. (12) Koepke, J.; Scharff, E. I.; Lucke, C.; Ruterjans, H.; Fritzsch, G. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1744-1754. (13) Hartleib, J.; Geschwindner, S.; Scharff, E. I.; Ruterjans, H. Biochem. J. 2001, 353, 579-589.
Scheme 1. Synthetic Route to O,O-Dialkylphosphoroamidates Employing the Todd-Atherton Reaction
very recent investigations have shown that this proposed mechanism is probably incorrect, as a number of H287 mutants still displayed substantial activity.14 Examples include H287F and H287L that show specific activities of 154 U/mg and 124 U/mg compared to the wild type activity of 194 U/mg. Water activation by these newly introduced amino acid residues is clearly not possible. Since the substrates for DFPase are rapidly hydrolyzed, attempts to characterize substrate binding to DFPase have not been possible to date. As substrate analogues acting as enzyme inhibitors and their structural characterization are indispensable tools for studying enzyme reaction mechanisms, we sought to design inhibitors of DFPase and investigate their interactions with the protein. We report the de novo design and synthesis of a DFPase substrate analogue that functions as a competitive inhibitor and the 1.73 Å crystal structure of the inhibitor O,Odicyclopentylphosphoroamidate bound to DFPase. We further characterize its binding mode in the active site through a combination of NMR, kinetic, and computational techniques. On the basis of these data, we propose an alternative reaction mechanism, involving calcium coordinating residue D229 as a nucleophile and a phosphoenzyme intermediate which existence is proven by 18O labeling experiments. Materials and Methods Docking. Docking studies were carried out employing the software AUTODOCK315 with low energy conformers of the ligands obtained with the CORINA program.16 Ligand Synthesis. The synthetic route to dialkylphosphoroamidates is straightforward using the dialkylphosphite17 as an intermediate with a subsequent Todd-Atherton reaction18 to the dialkylphosphoroamidate (Scheme 1). (Supporting Information) Expression and Purification of DFPase. DFPase was expressed and purified according to previously published procedures1 and is described in detail in the Supporting Information. (14) Katsemi, V.; Lucke, C.; Koepke, J.; Lohr, F.; Maurer, S.; Fritzsch, G.; Ruterjans, H. Biochemistry 2005, 44, 9022-9033. (15) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639-1662. (16) Sadowski, J.; Gasteiger, J. Chem. ReV. 1993, 93, 2567-2581. (17) Teichmann, B. J. Prakt. Chem. 1965, 4. Reihe, 94-98. (18) Atherton, F. R.; Openshaw, H. T.; Todd, A. R. J. Chem. Soc. 1945, 660663. J. AM. CHEM. SOC.
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ARTICLES Table 1. Enzyme Kinetics and Inhibition
Km (mM) kcat (s-1) kcat / Km (M-1 s-1) Ki (µM) D229N/N120D D229N/N175D E21Q/N120D E21Q/N175D
WT
WT + DcPPA (1 mM)
WT + DcPPA (3 mM)
2.72 ( 0.05 2107 ( 9 0.77 × 106
23.36 ( 0.07 2111 ( 7 0.09 × 106
65.45 ( 0.13 2091 ( 14 3.19 × 105
